Project Summary Molecular motors drive the active transport of organelles along the cellular cytoskeleton. This transport is critically important in neurons, highly polarized cells that extend axons up to 1m. Axons are continuously supplied with newly synthesized proteins and organelles from the cell body; active clearance of aging proteins and dysfunctional organelles is also required to maintain axonal homeostasis. Thus, axonal transport driven by the coordinated activities of cytoplasmic dynein and kinesin motors is essential, and deficits in this transport cause neurodegeneration. Here we focus on the molecular coordination of dynein and kinesin motors during axonal transport by scaffolding proteins and effectors, and the upstream regulatory kinases and phosphatases that maintain a sustained regulatory state over long length- and time-scales. We are also interested in interactions between microtubule- and actin-based motors, which affect both the initiation and termination of motility. Finally, we are interested in the mechanisms by which molecular motors and cytoskeletal dynamics actively remodel organelle membranes, leading to deformation, tubulation, fission and fusion. We will tackle these questions using the synergistic approaches of live cell imaging and in vitro reconstitution with single molecule resolution to understand the mechanisms involved. We will focus on three major goals. Goal 1: Understanding the integrated regulation of organelle transport. Each type of organelle moving along the axon has a distinct pattern of motility that directly relates to its function, but we do not yet fully understand the mechanisms regulating this transport. We will focus on essential axonal cargos, autophagosomes and signaling endosomes, testing the model that the cargo-specific, integrated regulation of motors allows for sustained transport over long time scales and distances. In Goal 2, we seek to understand the localized regulation of organelle dynamics within defined axonal zones, including the axon initial segment, presynaptic sites, and the axon terminal. These zones exhibit distinct trafficking patterns that correspond to differences in cytoskeletal organization: microtubule bundling, plus-end dynamics, post-translation modifications of tubulin, and intersections with actin filaments. We are interested in mechanisms that enhance the rate-limiting step of transport initiation, mediate compartment-specific sorting, and control cargo delivery/retention at specific sites of cellular need. And in Goal 3, we will study organelle remodeling driven by opposing motors and/or cytoskeletal dynamics. While some organelles move through the cell with little evident change in morphology, other cargos are dramatically remodeled, undergoing tubulation, fission or fusion. We hypothesize that molecular motors and cytoskeletal filaments provide an adaptable toolbox that can be specifically tuned to regulate dynamic organelle morphology. Together, these approaches should provide important new insights into organelle dynamics during axonal transport. As deficits in axonal transport lead to neurodegeneration, progress may provide new opportunities for targeted and effective therapeutic approaches.